Cells use selective degradation of misfolded and intrinsically unstable proteins to maintain physiologically appropriate levels of functional proteins. Imbalances between intracellular protein folding and degradation cause severe human diseases, via excessive degradation of mutated proteins (e.g. cystic fibrosis, retinitis pigmentosa), or toxic accumulation of misfolded proteins that overwhelms cellular degradation capacity (e.g. Alzheimer's disease). A key gap in understanding of protein folding-degradation relationships and mechanisms is its current restriction to only water-soluble (cytosolic) proteins, whereas little is known about membrane proteins, despite their major physiological and pathogenic importance. This knowledge gap is mainly due to inherent difficulties of analyzing folding of membrane proteins within their native lipid bilayer environment. Based on our strong preliminary data and novel steric-trapping molecular tools, the long-term objective of this project is to elucidate molecular mechanisms and determinants of membrane protein degradation, by defining the molecular and quantitative relationships between intrinsic folding properties of membrane proteins, including global vs. local stability, unfolding rates, and hydrophobicity of transmembrane segments, and their degradation. We will use an innovative combined model consisting of the membrane- integrated ATP-dependent E. coli protease FtsH as model degradation machine, and the intramembrane protease GlpG from E. coli as model substrate, both of which are widely conserved in prokaryotic and eukaryotic cells. The aims are: 1) To develop new methodologies for determining the stability of membrane proteins in their native bilayers, by adapting our prior steric trapping innovations, supported by preliminary findings. 2) Because substrate unfolding is a prerequisite to degradation mediated by ATP-dependent proteases, using these new methods, we will elucidate how the perturbation of GlpG structure caused by the force generated by FtsH-mediated ATP hydrolysis drives its unfolding, including cooperativity mechanisms, and identify the conformation of the resultant unfolded state that is targeted for degradation. 3) To define the quantitative relationship between folding and degradation rates of GlpG, including the influences of conformational stability and hydrophobicity, by analyzing degradation in a series of variants. Outcomes of this research will provide quantitative methods for analyzing membrane protein folding, will advance fundamental understanding and current concepts of cellular quality control systems for membrane proteins based on their folding properties, and will inform progress towards new therapies for diseases caused by aberrant protein- folding.